Leukotrienes are lipid-derived cell mediators that are released in response to a variety of immunologic and inflammatory stimuli. They are products of arachidonic acid metabolism derived through the 5-lipoxygenase pathway. Briefly, the initial step in leukotriene production involves oxygenation of arachidonic acid to produce 5S-hydroperoxy-6,8-trans-11,14 cis-eicosatetraenoic acid (5-HPETE), a subsequent dehydrase step producing the epoxide intermediate, 5,6-trans-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid (LTA4). Two routes of metabolism from LTA4 lead to the production of biologically active products. One of these pathways involves conjugation of LTA4 with glutathione (GSH) via LTC4 synthase to produce the sulfur-containing leukotriene 5S-hydroxy-6R-S-glutathionyl-7,9-trans-11,14 cis-eicosatetraenoic acid (LTC4). It is generally believed that LTC4 synthase is a member of the glutathione S-transferase enzyme family.
LTC4 has been implicated in a wide variety of diseases and pathologic conditions. LTC4 has been identified in fluids from psoriatic lesions and bronchial secretions associated with adult respiratory distress syndrome and neonatal pulmonary hypertension (for review, see Lewis et al., New Engl. J. Med., 323: 645, 1990, incorporated herein by reference).
Although the other enzymatic members of the 5-lipoxygenase pathway have been cloned, the cloning of LTC4 synthase has been problematic. This is partly because the synthase is very labile in partially purified form and because the endogenous production of LTC4 synthase in normal human cells is extremely small. LTC4 synthase is present only in limited types of normal human cells, namely granulocytes derived from bone marrow. Moreover, oligonucleotides developed from the N-terminal region of the LTC4 synthase polypeptide have not been specific enough to develop an effective screen because the N-terminal region is highly degenerate. In addition, an effective immunoassay for LTC4 which relies on incubation of substrate, has also been problematic since breakdown products of the substrate have been shown to cross-react with antibodies used in the assay.
It has already been established that inhibitors of 5-lipoxygenase and of the cell receptors for leukotrienes are of substantial efficacy in the management of patients with bronchial asthma. Given that are only three points at which the leukotriene metabolic system can be disrupted: the activation and function of 5-lipoxygenase; the receptor for the leukotriene; or the function of LTC4 synthase; characterization of LTC4 synthase would be important, notwithstanding the problems associated with its cloning.
Human leukotriene C4 synthase (also referred to herein as xe2x80x9cLTC4 synthasexe2x80x9d) has been cloned in an expression cloning system using a highly sensitive assay for LTC4, the product of the reaction catalyzed by LTC4 synthase. According to one aspect of the invention, an isolated nucleotide sequence encoding an LTC4 synthase polypeptide or unique fragments of human LTC4 synthase polypeptide, is provided. One embodiment is an isolated DNA sequence encoding a human LTC4 synthase polypeptide that has three hydrophobic transmembrane domains. Additionally, the invention relates to mammalian LTC4 synthase nucleotide sequences isolated from murine, porcine, ovine, bovine, feline, equine, or canine, as well as primate (e.g., simian) sources. Another embodiment is a human LTC4 synthase genomic clone, or unique fragments thereof.
Also provided are recombinant cells and plasmids containing the foregoing isolated DNA, preferably linked to a promoter. Portions of the foregoing nucleotide sequences are also included in the invention. One such portion is contained in a vector within a host cell.
According to another aspect of the invention, isolated human LTC4 synthase polypeptide is provided, having three hydrophobic transmembrane domains. Portions of the foregoing isolated human LTC4 synthase polypeptides are also included in the invention. Antibodies with selective binding specificity for the polypeptides of the invention also are provided.
Another aspect of the invention is a method for producing human LTC4 synthase polypeptide. The method includes providing an expression vector to a host, the vector containing a DNA sequence of the invention encoding for human LTC4 synthase polypeptide, allowing the host to express the human LTC4 synthase polypeptide, and isolating the expressed polypeptide.
A further aspect of the invention is an isolated nucleotide sequence capable of hybridizing to a target nucleotide sequence encoding human LTC4 synthase polypeptide. The target includes a nucleotide sequence encoding a human LTC4 synthase polypeptide with three transmembrane domains. The nucleotide sequence also can encode a human LTC4 synthase polypeptide having amino acid sequences unique to the polypeptide.
The novel molecules of the invention can be employed in experimental or therapeutic protocols. For example, a method for interfering with the activity of a human LTC4 synthase gene is provided, in which a construct is arranged to include a human LTC4 synthase nucleotide sequence that, when inserted into the genome of a cell, either inactivates transcription of messenger RNA for human LTC4 synthase polypeptide and/or inactivates translation of messenger RNA into human LTC4 synthase polypeptide in that cell. This construct further has a promotor operatively linked to the LTC4 synthase sequence. Next, the construct is introduced into a cell, and the construct is allowed to recombine with complementary sequences of the cell genome. Finally, cells lacking the ability to express human LTC4 synthase polypeptide are selected.
A further aspect of the invention is an assay method for identifying a modulator of a human LTC4 synthase polypeptide. One embodiment of the method includes providing a target cell containing an isolated nucleotide sequence which encodes for a human LTC4 synthase polypeptide. The target cell is maintained under conditions and for a time sufficient for the synthase to be expressed in the target cell. The target cell is then exposed to a compound suspected of modulating human LTC4 synthase polypeptide activity and a property of the target cell is measured in the presence of the modulator. This property is also measured in an identical target cell in the absence of the modulator. An altered property of the target cell exposed to the modulator is indicative of a modulating effect of the compound.
An alternative embodiment of the method of identifying LTC4 synthase modulators involves fusing LTC4 synthase gene promoter sequences (from the LTC4 synthase genomic clone) to a reporter gene, altering at least a portion of the promoter sequences, and detecting the effects of the alterations on expression of the reporter gene so that transcriptional regulatory sequences are identified. Yet another embodiment involves incubating fragments of the LTC4 synthase genomic clone with extracts of cells in which the LTC4 synthase gene is expressed and thereby identifying within those extracts factors that bind to LTC4 synthase genomic sequences and regulate LTC4 synthase gene expression.
A highly sensitive assay for LTC4, the product of the reaction catalyzed by LTC4 synthase, is also described herein. The assay includes steps of contacting a carrier having bound to it an amount of an LTC4 analogue (e.g., LTC2) and incubating the carrier in the presence of a solution containing an unknown amount of LTC4 synthase. Next, the carrier and solution are contacted with an amount of anti-LTC4 antibody under conditions and for a time sufficient for the anti-LTC4 antibody to bind with LTC4 in solution and with analogue (LTC2) on the carrier. Unbound anti-LTC4 antibody is separated from the carrier and then the carrier is contacted with a second antibody linked to a fluorescent label under conditions and for a time sufficient for the second antibody to bind with anti-LTC4 antibody associated with the carrier. The unbound second antibody is separated from the carrier and cell surface fluorescence of the carrier is analyzed by flow cytometry.
The present invention also provides the genomic location of the human LTC4 synthase gene, as well as methods of identifying LTC4 synthase gene polymorphisms that correlate with bronchial asthma. Methods of diagnosing susceptibility to bronchial asthma by detecting asthma-associated polymorphisms in LTC4 synthase genomic sequences are also provided.